The centromere is a multi-functional chromosomal domain that plays a major role in cell proliferation by mediating both chromosome movement and sister chromatid association during cell division. The kinetochore is a highly complex macromolecular structure associated with centromeric heterochromatin. The kinetochore is responsible for establishing and maintaining connections with microtubules of the mitotic spindle.
The identification and molecular cloning of many proteins of the centromere-kinetochore complex, using autoimmune sera and biochemical fractionation, has provided the necessary reagents to investigate the biochemical structure and function of the complex. The collection of proteins thus identified can be separated into two classes, based on their distribution during various times of the cell cycle. One class, the DNA- or chromatin-binding proteins, CENP-A, B, and C, are constitutive centromere proteins, inasmuch as they can be detected throughout interphase, at discrete loci within the nucleus (presumably centromere chromatin), or localized within the centromere-kinetochore complex during mitosis. The other class, comprising well-characterized proteins such as INCENP and CENP-E belong to the facultative family of centromere-kinetochore proteins, because of the transient nature of their association with the kinetochore complex. In the case of the kinesin-like CENP-E protein, it accumulates to high levels in the cytoplasm of interphase cells and assembles onto kinetochores of prometaphase chromosomes after nuclear envelope breakdown.
Measures of cell proliferation, including direct counts of mitotic figures, thymidine labelling, bromodeoxyuridine incorporation, determination of nucleolar morphology and flow cytometry, have been found to be prognostic indicators for a variety of tumor types. In addition, several proliferation-associated nuclear antigens have been identified which have allowed the direct detection and quantitiation of cycling cells by indirect immunofluorescence. One of these, Proliferating Cell Nuclear Antigen (PCNA), is a 36 kDa protein first identified by autoantibodies associated with autoimmune disease (Miyachi et al., J. Immunol. 121: 2228-2234, 1978). This protein is an auxiliary protein of DNA polymerase delta, a nuclear protein that is present throughout the cell cycle but is maximally expressed in the Gl-S phase (Matthews et al., Nature 303: 374-376, 1984). Several reports indicate that PCNA expression is deregulated in tumor cells, making the use of this antigen as a marker of tumor cell proliferation problematic (Hall et al., J. Pathol. 161: 285-294, 1990). A second cell cycle related antigen recognized by the monoclonal antibody Ki-67 has also been used to identify proliferating cells (Gerdes et al., Int. J. Cancer 31: 13-20, 1983). Like Ki-67, K1-K1 is detected from the G1 boundary of the cell cycle through M-phase. However, in contrast to Ki-67, Ki-S1 can be detected in paraffin embedded tissues. One major limitation of all three proliferating cell antigens described above is that they are detected throughout a major portion of the cell cycle, making them of limited use when specific cell cycle information is desired.
We recently identified a novel .ca 400 kDa cell cycle-dependent kinetochore associated protein in human cells, designated CENP-F, using human autoimmune serum (Rattner et al., Cell Motility and the Cytoskeleton 26: 214-226, 1993). Immunofluorescence staining of CENP-F revealed that it was homogeneously distributed throughout the nucleus of HeLa cells in the G2 stage of the cell cycle, and then localized to the centromere region of the chromosomes at prophase after G2. Similar to CENP-E, CENP-F appeared to localize to the outer layer of the kinetochore and was subsequently found at the spindle midzone and within the intercellular bridge during the later part of mitosis. CENP-F was not detected during the G1 phase of the cell cycle, and was presumed discarded or degraded after cytokinesis.
Amongst the small number of facultative centromere-kinetochore proteins that have identified thus far, CENP-F appears to be the earliest member to interact with the centromere-kinetochore complex. For example, the appearance of CENP-F at the centromere-kinetochore region at prophase precedes the appearance of CENP-E. Thus, antibodies immunologically specific for CENP-F could enable the specific detection of G2 and M-phase cells in cultured cells or in tissues, rendering CENP-F a useful addition to the cell cycle specific markers used to study cell proliferation in tumor samples. However, to date, little information is available regarding the molecular characterization of CENP-F; the isolated purified CENP-F protein has heretofore been unavailable, and likewise, nucleic acids encoding the protein have not been produced.
It is an object of the present invention to provide an isolated, purified, nucleic acid molecule that encodes the entire CENP-F polypeptide. It is further an object of the invention to provide an isolated, purified form of the CENP-F protein, and to provide antibodies immunologically cross-reactive with the protein. The nucleic acid, protein and antibodies thereto may be used to advantage in diagnostic and prognostic assays for the detection and measurement of cellular proliferation in cultured cells and in tissues. The use of CENP-F as a cell cycle-specific marker advantageously allows the specific detection of G2 and M-phase cells, which other known cell cycle-specific markers are unable to specifically target. Additionally, the CENP-F protein itself provides the additional benefit of use for detecting autoimmune antibodies to the protein, which may provide an early diagnosis for the onset of various malignant diseases.